The term “immunotherapy” defines the treatment of diseases by inducing, enhancing or suppressing an immune response. The strategy of immunotherapies is to fight diseases, such as cancer, infectious diseases, allergy and asthma.
A variety of active agents, so called immunomodulators, that can be used in immunotherapy are known. Specific DNA sequences belong to those known immunomodulators. Most known immunomodifying short DNA sequences contain an unmethylated cytosine guanine motif (CG motif), which has been described by Krieg et al. (Nature 1995 374: 6522 546-549). The occurrence of unmethylated CG motifs is substantially suppressed in the genome of eukaryotes compared to prokaryotes or viruses. Therefore, DNA molecules containing such a motif have evolved as a natural “danger signal” and trigger the immune system in the fight against prokaryotic or viral pathogens. This can be exploited therapeutically or prophylactically by using such sequences to treat or prevent infectious with immunotherapy.
DNA constructs comprising unmethylated CG motifs are able to elicit a considerable physiological effect by strongly stimulating effector cells of the innate immune system including dendritic cells, macrophages, natural killer (NK) and NKT cells. Unmethylated CG motifs are detected by the innate immune pattern recognition receptor Toll-like receptor (TLR) 9. While the exact recognition mechanism is not yet fully understood, significant progress in unraveling the underlying pathways has been made (A. Krieg, Nat. Rev. Drug Disc., 5:471-484, 2006).
It is assumed that upon binding of DNA constructs containing unmethylated CGs to the receptor, multiple signal cascades are activated in responding cells. By upregulation of characteristic surface molecules and secretion of cytokines, adaptive immunity with a predominant Th1 pattern is induced. Such constructs can be used in combination with, for example, antibodies, chemotherapy or radiation therapy, vaccines or cytokines. Allergic diseases and asthma are mostly Th2-mediated. By increasing the ratio of Th1/Th2, the Th2-mediated responses are attenuated and thereby these types of diseases can be treated or prevented.
Surface molecules, which are upregulated by the TLR-9 pathway, include, for example, CD40, CD69, CD80, CD86 or CD169, depending on the cell type. The enhanced secretion of cytokines is also characteristic for distinct cell types; cytokines include, for example, macrophage inflammatory proteins (MIP)-1alpha, MIP-1beta, interleukin (IL)-6, IL-8, interferon (IFN)-alpha, tumor necrosis factor (TNF)-alpha, IFN-gamma, monocyte chemotactic protein (MCP)-1 or IFN-gamma-induced protein of 10 kDa (IP-10).
In order to prevent or treat diseases, vaccination has been proven as a very effective approach. To ensure a strong and durable immune response, adjuvants capable of stimulating antigen-presenting cells such as dendritic cells, are usually administered together with the antigen, and for that purpose TLR9 agonists have been shown to be potent immunostimulants.
Preclinical and ongoing clinical studies support the use of TLR-9 agonists as immunomodulators and/or adjuvants, and proof their anti-tumor effect by enhancing both the humoral and cellular responses.
Independently of any explanations of the underlying mechanisms by which unmethylated CG motifs influence or modulate an immune response, many approaches were developed for modulation of the immune system by using such motifs. The WO 1998/018810 discloses that immunostimulatory sequences containing unmethylated CG motifs are even more effective when they are part of a single strand. However, administering an open-chained single-stranded DNA molecule is not practicable due to the quick degradation of single-stranded nucleic acids. Consequently, different methods for the protection of single- or double-stranded DNA constructs comprising an unmethylated CG motif were developed.
To achieve resistance against the degradation by DNA nucleases the phosphodiester bonds in the backbone of a nucleic acid polymer are frequently modified to phosphorothioates. Besides a somewhat less stimulatory activity of such phosphorothioate-protected nucleic acids clinical trials within the last years showed that the toxicity of a phosphorothioate-protection exclude or severely limit such nucleic acids from any use in pharmaceutical compositions or medicaments.
From the 4 classes of known activators with distinct immunomodulation profiles all members except one comprise of linear DNA molecules. The exception is disclosed in EP 1 196 178. This document discloses short deoxyribonucleic acid molecules, comprising a partially single-stranded, dumbbell-shaped, covalently closed sequence of nucleotide residues comprising CG motifs (“dSLIM”) consisting entirely of natural DNA. According to the disclosure of the EP 1 196 178 the CG motifs are located within the single-stranded loops at both ends of the double-stranded stem of the disclosed molecule or within the double-stranded stem. The single-stranded hairpin loops protect a double-stranded stem from degradation by DNA nucleases within or outside of the cell. Document US 2009/0053250 A1 discloses such dumbbell-shaped, covalently closed molecules comprising CG motifs in combination with chemotherapeutic drugs. US 2007/0049546 A1 discloses the dumbbell-shaped, covalently closed molecules comprising CG motifs with covalently attached one or more substituents. The publication “Schmidt M. et al., 2006, “Cytokine and Ig-production by CG-containing sequences with phosphorodiester backbone and dumbbell shape”, Allergy, Vol. 61, pp. 56-63” suggests that such dumbbell-shaped, covalently closed molecules comprising certain CG motifs might be useful in the treatment of allergic diseases.
Document WO 2010/039137 discloses immune regulatory oligonucleotides as antagonists for TLR mediated diseases having one or more chemical modifications in the sequence flanking an immune stimulatory motif and/or in an oligonucleotide motif that would be immune stimulatory but for the modification. Thus, the intention of the disclosed oligonucleotides of WO 2010/039137 is to suppress an immune response caused by TLRs.
WO 2005/042018 describes new so-called C-class CpG oligonucleotides, wherein a c-class oligonucleotide is characterised by CpG sequences, generally positioned at or near the 5′ end or 3′ end of the molecule, and a GC-rich palindrome motif, generally positioned at or near the other end of the molecule. The document discloses variations of the palindromic sequence of a c-class DNA.
Above-cited publication “A. Krieg, Nat. Rev. Drug Disc., 5:471-484, 2006” discloses distinct classes of CpG oligodeoxynucleotides and their effects but does not disclose dumbbell-shaped, covalently closed molecules comprising CG motifs which avoid phosphorothioate modification.
WO 2004/000873 A2 also discloses phosphorothioate-modified CG-containing single-stranded DNA, which can be added to fusion peptides as an adjuvant. This document dose not disclose dumbbell-shaped, covalently closed molecules comprising CG motifs.
Likewise, US 2003/0050263 deals with CG-containing oligonucleotides in the context of the treatment of HIV infections but does not deal with dumbbell-shaped, covalently closed molecules comprising CG motifs in any sequence context.